Abstract

The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules that are coupled permanently to their targets. This issue prohibits characterization of important functional relationships between different molecular pathway components in cells. Yet, recent improved understandings of DNA strand displacement reactions now provides opportunities to create programmable labeling and detection approaches that operate through controlled transient interactions between different dynamic DNA complexes. We examined whether erasable molecular imaging probes could be created that harness this mechanism to couple and then remove fluorophore-bearing oligonucleotides to and from DNA-tagged protein markers within fixed cell samples. We show that the efficiency of marker erasing via strand displacement can be limited by non-toehold mediated stand exchange processes that lower the rates that fluorophore-bearing strands diffuse out of cells. Two probe constructions are described that avoid this problem and allow efficient fluorophore removal from their targets. With these modifications, we show one can at least double the number of proteins that can be visualized on the same cells via reiterative in situ labeling and erasing of markers on cells.

Labeling and removal of Cy5 fluorophores from protein markers via strand displacement reactions of a two-strand probe complex (PC2s). (A) Selective labeling of expressed GFP proteins in CHO cells. The images display a strong correspondence between the GFP and two-strand probe (Cy5) signals; pixel intensities of the GFP and Cy5 signals are linearly correlated (r2 = 0.94). However, OFF signal intensities indicate ∼20% of active Cy5 dye remains on the cells after the erasing reaction. (B) Pixel intensities for cross section indicated in the probe images in A for both the ON and OFF states of the cells. (C) Histogram of the average Cy5 signal intensities for the ON and OFF states of 20 cells. Cells are grouped based on their GFP whole-cell fluorescence intensities (low, medium and high levels correspond to 2000–5500, 5500–10 000 and 10 000–15 800 intensity units, respectively).

Labeling and removal of Cy5 fluorophores from protein markers via strand displacement reactions of a three-strand probe complex (PC3s). (A) Selective labeling of expressed GFP proteins in CHO cells. The OFF reactions are now efficient, and yield a signal (cell intensity) to background (slide surface intensity) ratio of 1.08. The ratio of labeled/erased probe intensities, or ON/OFF ratio, is 28.6. This result is reflected in (B) pixel intensities for cross sections as well as (C) histogram of average, whole-cell Cy5 signal intensities for 20 cells in their ON and OFF states.

Kinetics of DNA strand displacement reactions on fixed cells. (A) Labeling and erasing reactions of a two-strand probe complex (PC2s). Each curve represent the average intensity of an individual cell within the sample. The erasing reactions are inefficient and significant signals remain on the sample even after a 2-h incubation period. (B) Labeling and erasing reactions of a three-strand probe complex (PC3s) showing rapid and efficient erasing. The arrows in each plot indicate the time point where the labeling reactions were stopped and the erasing reactions were initiated.

Labeling of DNA-tagged GFP proteins through non-toehold mediated exchange via a reaction of TS with a quenched W complex as a probe. The concentration of the W complex was 200 nM, and the reaction was performed without adding E to the reaction mixture.

Multiplexed (multi-color) and reiterative (sequential) labeling of four different TS strands coupled to the GFP-ZE targets at equimolar ratios. The scheme at the left depicts the labeling steps for each set of probe images.